Toxicity Evaluation of a Polyphenolic Extract from Flourensia cernua DC through Artemia Lethality Assay, Hemolytic Activity, and Acute Oral Test

Flourensia cernua DC, commonly known as hojasen or tarbush, is a medicinal plant used in arid regions due to its therapeutic properties, especially in the treatment of gastrointestinal disorders. This study aimed to assess the toxicity of a polyphenolic extract obtained from F. cernua. This research involved both in vitro (hemolytic and brine shrimp assay) and in vivo tests (acute oral toxicity) to determine the safety profile of this extract. The extract was obtained through a novel ultrasound-microwave extraction and purified by ion-exchange chromatography. Analysis of the polyphenolic extract revealed a rich composition of flavonoids and hydroxycinnamic acids, mainly apigenin glycosides. In toxicity tests, the polyphenols did not exhibit toxicity towards Artemia salina at a concentration of 1 mg/ml. Furthermore, incubation at 500 μg/ml for 4 hours showed a slight toxic effect on erythrocytes. In the acute oral toxicity test in mice, doses of 300 mg/kg and 2000 mg/kg did not result in animal mortality, indicating that the LD50 exceeds 2000 mg/kg. However, the higher dose induced signs of toxicity, including lethargy, drowsiness, piloerection, and a significant decrease in weight during the initial two days postadministration of the polyphenolic extract. In addition, histological analysis suggested potential kidney damage at the 2000 mg/kg dose. According to OECD guidelines, while the extract can be classified as category 5 (low acute toxicity) due to the absence of mortality at 2000 mg/kg, the observed signs of toxicity should be considered in the overall risk assessment. These findings highlight the potential of F. cernua in pharmaceutical and nutraceutical applications due to its high polyphenolic content. However, further investigations are necessary to explore the specific effects of the compounds present in the extract. In addition, continuous evaluation of its long-term toxicity is essential to fully understand the extract's safety profile and efficacy.


Introduction
Te northern regions of Mexico, with their semiarid climate, host a diverse array of wild plants that thrive under extreme climatic conditions [1].One such abundant plant in the Chihuahuan desert is Flourensia cernua.Tis region spans parts of Mexican states, Chihuahua, Coahuila, Durango, Zacatecas, San Luis Potosí, Nuevo León, and Tamaulipas, as well as Arizona, New Mexico, and western Texas in the United States of America [2].F. cernua, commonly known as hojasen, tarbush, blackbrush, varnish bush, hojase, hoja ancha, and arbusto de alquitrán.It is a shrub characterized by its light brown or gray stem, which typically grows around 1 meter in height [3,4].Te shrub is highly branched, resinous, and emits a distinctive tar-or hop-like odor.Its leaves are alternate, ovate to oval, and dark green [5,6].Te plant bears pendulous fowers containing several yellow disc forets, and its fruit is a hairy achene, measuring up to 1 cm in length [7].
In Mexican markets, F. cernua leaves and fowers are commercially available for preparing infusions and are used in traditional medicine.Notably, F. cernua leaves have been used as a remedy for various gastrointestinal ailments, such as diarrhea, indigestion, and stomach pain.Te plant has a history of traditional use because of its purgative, astringent, expectorant, and antirheumatic efects [5,8,9].Te phytochemical composition of F. cernua includes an array of polyphenolic compounds and essential oils.Mainly, favonoids, such as favones, favanones, and favonols, have been identifed as the main constituents [10,11].Te tarbush resin contains sesquiterpenes, acetylenes, p-acetophenones, benzofurans, and benzopyrans.Recent studies have explored the biological properties of the plant, and its extracts have been associated with antioxidant, anti-infammatory, anticancer, and antidiabetic activities in vitro [12,13].Given its potential as a source of pharmacologically active compounds, evaluating the safety of its constituents is crucial.Some studies have reported the toxicity of F. cernua; its dry fruits have been described as toxic when consumed at approximately 1% of the animal's body weight, potentially leading to fatalities within the frst 24 hours [14].Chronic consumption of its leaves has been associated with liver damage, evidenced by elevated levels of serum gamma-glutamyl transpeptidase and aspartate aminotransferase activities, as well as hepatocytes apoptosis in ewe lambs [15].Furthermore, the administration of a single 2000 mg/kg oral dose of tarbush extract resulted in signs of toxicity such as lethargy and drowsiness in rats within half an hour [16].Terefore, this study aims to identify and quantify the polyphenol content in a hydroethanolic extract of F. cernua leaves.It also seeks to evaluate its toxicity using brine shrimp, hemolytic activity, and acute oral toxicity tests in mice.

Materials and Methods
2.1.Vegetal Material.Flourensia cernua DC samples were collected from the "La Angostura" region in Coahuila de Zaragoza (latitude: 25.30909 and longitude: -101.07395) in August.Random bushes were selected, and their branches were pruned (Figure 1).A specimen was pressed and prepared for identifcation at the ANSM herbarium, located in the Department of Botany at the Antonio Narro Agrarian Autonomous University.Professor José A. Villarreal Quintanilla assigned it the registration number 104702.Te leaves were then air-dried at room temperature (22-26 °C) for 72 h.After drying, the branches were separated, leaving only the leaves for further processing.Subsequently, the leaves were ground and sieved using a RO-TAP (Test Sieve Shaker, W.S. Tyler ™ ), and particles smaller than 0.6 mm were collected.

Extraction of Phytochemical Compounds.
A hydroethanolic extract of F. cernua leaves was obtained using a mass: volume ratio of 1 : 12. Initially, 83.33 g of the dry sample was weighed and mixed with 1 L of 30% ethanol.Te mixture was then subjected to an ultrasonic-microwave (US-MW) reaction system (Nanjing ATPIO Instruments Manufacture Co. Ltd., Nanjing, China) under the conditions described in previous research [17].Te resulting hydroethanolic extract was fltered three times using flter paper (Whatman No. 41) to remove insoluble material and then stored at 4 °C in amber containers.

Purifcation of Polyphenolic Compounds.
After extract preparation, polyphenols were purifed using ion-exchange chromatography, employing AmberLite XAD-16 (Milli-poreSigma) as the stationary phase and a mobile phase consisting of water followed by 96% ethanol.Initially, water was passed through the column to wash away nonpolyphenolic compounds.Ten, 96% ethanol was used as the desorption solvent to elute the polyphenols from the AmberLite resin.Te ethanolic solution was collected in an Erlenmeyer fask until the mobile phase no longer exhibited color, indicating that the elution of polyphenols was complete.A 1 : 1 ratio of ethanol to extract volume was used to ensure complete elution of the polyphenols.Subsequently, the drying process was conducted in an oven set at 50 °C for 24 h to evaporate the ethanol.Once dried, the polyphenols were scraped with a stainless-steel spatula to recover the powder, which was then weighed and stored in amber bottles at room temperature [18].

Determination of Total Hydrolyzable Polyphenols.
To quantify the total hydrolyzable polyphenols, the Folin-Ciocalteu method was employed [19].A 15 μl sample of the polyphenol fraction at a concentration of 0.5 mg/ml (dissolved in 96% ethanol) was placed and mixed with 170 μl of Milli-Q water in a 96-well microplate in triplicate.Next, 12 μl of Folin-Ciocalteu reagent (Millipore) was added, and the mixture was incubated in the dark for 5 min at room temperature.Subsequently, 30 μl of Na 2 CO 3 (200 g/L) was added, and the solution was further incubated in the dark for one hour at room temperature.Finally, 73 μl of water was added, and the absorbance was read at 765 nm using a spectrophotometer (Epoch BioTek ELISA plate reader).A calibration curve with gallic acid (Sigma-Aldrich; G7384) was used as the reference standard.Te results were expressed as milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g).

Determination of Condensed Polyphenols.
Te quantifcation of condensed polyphenols was performed using the HCl-butanol method [18].In a 1.5 ml microtube, 167 μl of the sample (1 mg/ml), 1 ml of HCl-butanol (1 : 9), and 33 μl of ferric reagent (2% ferric ammonium sulfate in 2 N HCl) were combined.Te mixture was then heated at 100 °C in a digital dry bath (Labnet D1100) for 1 h.After cooling, 300 μl from each tube was transferred to a 96-microplate well in triplicate.Finally, the absorbance at 460 nm was measured using a spectrophotometer (Epoch BioTek ELISA plate reader).Results were expressed as milligrams of catechin equivalent per gram of dry extract (mg CE/g) based on a calibration curve with catechin (Sigma-Aldrich; C1251).

Determination of Flavonoid Content.
Te quantifcation of favonoids was conducted based on their reaction with aluminum ions in a basic medium [20].In a 1.5 ml microtube, 200 μl of the sample (1 mg/ml) was mixed with 280 μl of Milli-Q water and 60 μl of 5% NaNO 2 , and the resulting mixture was thoroughly blended.Te solution was then incubated for 5 min at room temperature.Afterwards, 60 μl of 10% AlCl 3 was added, mixed, and incubated for 10 min at room temperature.Finally, 400 μl of 1 M NaOH was added to the mixture.From this, 200 μl was placed in a 96-microplate well in triplicate.Te absorbance was read at 510 nm using a spectrophotometer (Epoch BioTek ELISA plate reader).Te results were expressed as milligrams of catechin equivalent per gram of dry extract (mg CE/g) based on a calibration curve with catechin (Sigma-Aldrich; C1251).

Identifcation of Polyphenolic Compounds by HPLC/MS.
Characterization of the F. cernua polyphenolic extract was carried out using reversed-phase high-performance liquid chromatography (HPLC) with a diode array detector (280 nm) and a mass spectrometer (MS) equipped with a liquid chromatography ion trap (Varian 500-MS IT; Agilent, Santa Clara, CA, USA) and an electrospray ion source.One milligram of the sample was diluted in 1 ml of 96% ethanol, fltered through a 0.45 μm nylon membrane, and 5 μl were injected into a Denali C18 column (3 μm, 150 mm × 2.1 mm; Grace, Albany, OR, USA) maintained at 30 °C.Eluents consisted of formic acid (0.2%, v/v; solvent A) and acetonitrile (solvent B).Te elution conditions were in accordance with a previous study [21].Mass spectrometer experiments were performed in negative mode [M-H] −1 .Data were collected and processed using the MS Workstation software (version 6.9; Agilent, Santa Clara, CA, USA).Te samples were frst analyzed in a full scan mode acquired in the m/z range 50-2000, and further analyses were performed on a series of selected precursor ions.Te HPLC/MS analyses were conducted using a bioactive compounds database (WorkStation version 2.0 database; Varian, Palo Alto, CA, USA).To estimate the relative content of the identifed polyphenols, the area of each peak was calculated using the software GraphPad Prism 6.

Toxicity in Brine Shrimp
. Te toxicity test in Artemia salina was conducted following previously reported methods [22].Initially, 83 mg of brine shrimp cysts were placed in 500 ml of simulated seawater (Supplementary Table 1) and kept at 26 °C with intense light (5500 lux) in an artifcial climate chamber (ECOSHEL C268D) for 24 h to promote cyst hatching.For the assay, 1900 μl of simulated seawater was dispensed into each well in a 24-well microplate and 10 brine shrimp nauplii were added to each well.Subsequently, 100 μl of the polyphenolic extract of F. cernua was placed at 5 diferent concentrations, 50, 100, 200, 500, and 1000 μg/ml, in triplicate.As a positive toxicity control, potassium dichromate (K 2 Cr 2 O 7 ) was used at a concentration of 200 μg/ ml.Polyphenolic extracts at diferent concentrations were prepared using a vehicle composed of 5% Tween 80, 5% DMSO, and 90% phosphate-bufered saline (PBS).In addition, a negative control with simulated seawater and a vehicle control were included.Following the addition of treatments, the brine shrimp nauplii were incubated for 24 h at 26 °C in the dark.After incubation, the brine shrimp nauplii in each well were counted, considering a brine shrimp dead if it remained motionless for 10 s.Finally, the percentage of mortality for each treatment was calculated according to the following equation: % mortality � total Artemia salina − living Artemia salina total Artemia salina   * 100. (1) 2.9.Hemolysis Assay.Te hemolysis assay was performed on two types of blood samples: mouse blood and human blood.
Te mouse blood sample was obtained from female mice of the BALB/c strain, acquired from the School of Chemistry, Autonomous University of Coahuila.Te blood from mice was collected by intracardiac puncture using an EDTA (10%)-impregnated syringe with a 27G × 13 mm needle.Before the procedure, all animals were anesthetized with pentobarbital (150 mg/kg).Te human blood sample was obtained from a healthy volunteer with no previous medical history of chronic diseases.Phlebotomy was performed on the peripheral veins of the arm, using a Vacutainer ® 21G × 38 mm needle and a BD Vacutainer ® K2 EDTA tube.
After collection, the blood samples were centrifuged at 600 g for 5 min.A line was marked on the tube at the same height as the supernatant, which was then removed.Te same Journal of Toxicology volume of PBS solution was added to the erythrocyte suspension, carefully mixed, and centrifuged again at 600 g for 5 min.Te supernatant was removed, and the washing process with the PBS solution was repeated twice.Once the washing steps were completed, the erythrocytes were suspended in PBS at a concentration of 2% (v/v).For this, 10 μl of the erythrocyte pellet was diluted in 480 μl of PBS with 10 μl of the polyphenolic extract of F. cernua at diferent concentrations (100, 200, and 500 μg/ml).Te polyphenolic powder was dissolved in two vehicles as follows: (a) 5% Tween 80, 5% DMSO, and 90% PBS and (b) 25% DMSO and 75% PBS.Te samples were then incubated at 37 °C for 1, 2, 3, and 4 h.After incubation, the samples were centrifuged at 2000 g for 5 min and 100 μl of the supernatant was placed in a well of the 96-well microplate in triplicate.Finally, the samples were read at a wavelength of 540 nm using a spectrophotometer Epoch BioTek ELISA plate reader.PBS and distilled water were used as negative and positive controls, respectively.Te equation used to calculate the percentage of hemolysis was as follows: Before the experiment, the mice were habituated in individual cages for one week.Te assay began with a test dose of 300 mg/kg of the polyphenolic extract of F. cernua (n � 6); this is due to OECD guidelines 423, since it is recommended to use the starting dose of 300 mg/kg of body weight when there is no information about the substance to be tested.Mice were fasted for 4 h, allowing only access to water before administering the polyphenolic extract.Ten, the extract, dissolved in the vehicle (5% DMSO, 5% Tween 80, and 90% PBS), was administered orally with an oral gavage needle (20G × 38 mm).After that, the food was removed for an additional 2 h.Animals were observed periodically during the frst 24 hours, with particular attention given to the frst 4 hours, and then daily for a total observation period of 14 days.Mice were weighed daily throughout this period.If no mortality was observed in mice treated with the 300 mg/kg dose, an additional group of mice was used at a higher dose of 2000 mg/kg (n � 6).For the 2000 mg/kg dose, the administration was divided into two 1000 mg/kg doses with a period of 4 hours between each administration.Similarly, mice were closely observed for the frst 24 h and monitored for 14 days.In addition, a group of mice (n � 6) that received only the vehicle orally was used as the control.At the end of the observation time, mice were euthanized with sodium pentobarbital (150 mg/kg).A dissection was performed, and blood was obtained by intracardiac puncture.Te liver, kidneys, and brain were then obtained and fxed in 4% formaldehyde for 48 h.
2.12.Statistical Analysis.Data were processed using the GraphPad Prism statistical program version 6.01 (San Diego, CA, USA).For the toxicity in brine shrimp, a one-way ANOVA with Tukey's post hoc test was used.For the hemolysis assays and the weights obtained in the acute oral toxicity, a two-way ANOVA followed by Tukey's multiple comparison test was applied.Te results were expressed in mean ± standard error of the mean (SEM), and a p value of ≤0.05 was considered statistically signifcant.

Polyphenol Profle of F. cernua.
Te quantifcation of hydrolyzable and condensed polyphenols, alongside favonoids, was conducted in a polyphenolic-enriched extract obtained after AmberLite ion-exchange chromatography.Results indicated a higher concentration of hydrolyzable polyphenols compared to condensed tannins.In addition, the extract displayed a favonoid content of 124.8 ± 6.9 mg CE/g (Table 1).

Toxicity in Brine
Shrimp.To evaluate the potential toxicity of F. cernua polyphenols, the lethality assay was performed in brine shrimp (Figure 2).Te vehicle used to dissolve the polyphenols did not afect the viability of brine shrimp, while the positive control (K 2 Cr 2 O 7 ) resulted in a 100% mortality rate.Te toxicity tests in brine shrimp revealed that the polyphenolic extract of F. cernua exhibited a maximum mortality percentage of 14.4 ± 3.4 at the highest concentration of 1000 μg/ml used in the test, which was signifcantly diferent from the other concentrations (p ≤ 0.001).Concentrations of 50 and 100 μg/ml did not cause the death of any brine shrimp nauplii.Concentrations of 200 μg/ml and 500 μg/ml resulted in a mortality rate of 2.2 ± 1.5% and 4.4 ± 1.8%, respectively.In addition, all concentrations of the F. cernua extract used showed a lower % mortality compared to the positive control (p ≤ 0.001).

Hemolysis Assay.
In the hemolysis assay using mouse and human erythrocytes, various vehicles were tested to dissolve F. cernua polyphenol powders.Te combination of Tween 80, DMSO, and PBS caused lysis of mouse erythrocytes at all incubation times in a similar way to the positive control used (data not shown).Consequently, a solution of F. cernua extract dissolved in a 25 : 75 ratio of DMSO and PBS was used, showing no hemolysis during any incubation period.Concentrations of 100 and 200 μg/ml caused no hemolysis in mouse erythrocytes at any time.However, at 500 μg/ml, hemolysis occurred after the third hour compared to the other concentrations (p ≤ 0.05), reaching a hemolysis rate of 7.0 ± 3.1% in the third hour and 5.7 ± 2.5% in the fourth hour (Figure 3).In hemolysis tests with human erythrocytes, using polyphenolic extract of F. cernua diluted in Tween 80, DMSO, and PBS (Figure 4), no hemolysis was observed during the frst two hours of incubation at any concentration evaluated.In the third hour, concentrations of 100, 200, and 500 μg/ml showed slight hemolysis (3.1 ± 0.8, 5.1 ± 0.8, and 2.1 ± 0.1, respectively), without signifcant diferences.
Te comparison between the polyphenolic extract of F. cernua and its vehicle revealed a cytoprotective efect on human erythrocytes, as evidenced by a lower level of hemolysis compared to the vehicle.In the frst two hours of incubation, the vehicle (Tween 80, DMSO, and PBS) did not induce hemolysis in red blood cells.However, in the third hour, a signifcant increase in hemolysis was observed in vehicle-treated erythrocytes (21.0 ± 0.3%) compared to those treated with the polyphenolic extract of F. cernua dissolved in the vehicle (p ≤ 0.001).By the fourth hour, hemolysis induced by the vehicle notably increased to 56.4 ± 1.1%, with a signifcant diference from erythrocytes treated with the polyphenols (p ≤ 0.001).Among the extract concentrations, the 100 μg/ml concentration showed the lowest percentage of hemolysis compared to the others (p ≤ 0.001).
Te evaluation of % of hemolysis in human erythrocytes using the extract of F. cernua dissolved in the vehicle constituted by DMSO: PBS (25 : 75) showed that concentrations of 100 and 200 μg/ml maintained hemolysis values below 5% during the four hours of incubation (Figure 5).However, at 500 μg/ml, a hemolytic efect was observed, escalating over time, and reaching 28.7 ± 1.2% at the fourth hour (p ≤ 0.001).Tese results indicate that polyphenolic extract of F. cernua at 100 and 200 μg/ml did not induce signifcant hemolysis on human erythrocytes.Nonetheless, the concentration of 500 μg/ml exhibited a dose-dependent hemolytic efect over time.

Acute Oral Toxicity.
Te acute oral toxicity test was performed following OECD 423 guidelines.Te oral administration of polyphenolic extract of F. cernua at doses of 300 and 2000 mg/kg did not result in mortality in mice.However, signs of toxicity were observed within the frst 24 h after dosing.At 300 mg/kg, mice exhibited lethargy and piloerection for 30 min after dosing but showed normal behavior thereafter for the next 24 h and for the entire 14 days of the test.
At the dose of 2000 mg/kg, mice exhibited lethargy, drowsiness, piloerection, reduced appetite, rapid breathing, and hunched posture during the frst two hours after extract administration.Te frequencies of occurrence of the signs in each experimental group are shown in Supplementary Table 1.Nevertheless, they gradually improved and showed normal behavior after 24 h of administration, which was maintained during the 14-day observation period.Te results showed a weight decline in mice administered 2000 mg/ kg within the initial 48 hours, but gradually regained their initial weight over the subsequent days (Figure 6(a)).When comparing group weights before and after administration (Figure 6(b)), a signifcant initial decrease was observed in the 2000 mg/kg group compared to the control (p ≤ 0.05) during the frst two days after administration.No signifcant hemolysis was observed at 1 and 2 hours of incubation compared to the vehicle.However, signifcant hemolysis ( * * p ≤ 0.01) was observed at 3 and 4 hours of incubation with the concentration of 500 μg/ml.Each treatment was performed in triplicate in two independent experiments.Statistical analysis was performed using a two-way ANOVA followed by Tukey's post hoc test.Data are presented as mean ± SEM. ns: not signifcant.
glomerular and renal tubular architecture.However, in the group administered with the higher dose of 2000 mg/kg of polyphenolic extract, a reduction in glomerular size and shrinkage was observed, indicating potential mild renal alterations at this higher dose.Analysis of brain tissue from all experimental groups did not show noticeable histological alterations in the hippocampus, cortex, and cerebellum region (Supplementary Figure 2).In summary, histopathological evaluation of liver, kidney, and brain tissues suggests that the administration of F. cernua polyphenolic extract, particularly at the higher dose, may induce mild changes in the kidneys without afecting liver and brain tissues.

Discussion
In regions with restricted healthcare access, such as remote arid zones, communities have traditionally relied on herbal remedies to address diverse diseases [23].Flourensia cernua, a plant thriving in the semidesert areas of Chihuahua, is commonly used in leaf infusions or decoctions to alleviate gastrointestinal ailments [24].Despite its traditional usage, comprehensive data on the compounds within its leaves and the potential associated toxicity after consumption remain limited.
In this study, we obtained a hydroethanolic extract of F. cernua using emerging ultrasound-microwave techniques, followed by purifcation of polyphenols via ion-exchange chromatography.Te polyphenolic extract showed a notably higher content of hydrolyzable polyphenols (257.7 ± 8.8 mg GAE/g) compared to condensed polyphenols (100.2 ± 13.2 mg CE/g).Tis observation aligns with previous fndings by Estell et al. [25], who also highlighted a predominance of hydrolyzable polyphenols in the plant compared to condensed polyphenols.In other studies, the hydrolyzable polyphenols of F. cernua were quantifed obtaining values of 301 ± 0.2 mg GAE/g and 53.07 mg GAE/g by the Soxhlet extraction method [5,12].Diferent extraction methods, solvents, soil conditions, nutrient availability, temperature, light intensity, harvest season, and degree of plant maturation can contribute to variations in polyphenol content [26,27].Te extract is rich in favonoids (124.8 ± 6.9 mg CE/g), which is in agreement with a study reporting quercetin equivalent concentration within the range of 258.22-133.77μg/ml [8], emphasizing, F. cernua leaves as a rich source of favonoid-like compounds.Te HPLC/MS analysis identifed 20 distinct compounds in the F. cernua extract, predominantly belonging to favonoids and hydroxycinnamic acids.Flavonoids such as favones, favanones, and favonols constitute the primary polyphenolic components of the Asteraceae family [9,28].Previous studies have also identifed compounds present in this plant.Álvarez-Perez et al. [10] and Aranda-Ledesma et al. [8]  Each treatment was performed in triplicate in two independent experiments.Statistical analysis was performed using a two-way ANOVA followed by Tukey's post hoc test.Data are presented as mean ± SEM. ns: not signifcant.* * * p ≤ 0.001.
apigenin and its glycosides were identifed as the primary compounds in the polyphenolic extract, which is consistent with prior research fndings.Te abundance of favones and other favonoids in F. cernua may be attributed to their role as UVB protectors in plants, as they absorb light within the 280-315 nm range.Due to the semidesert habitat and high UVB exposure of F. cernua, a high polyphenol content, especially favones, is expected [29,30].Apigenin, a widespread favonoid in plants, is commonly found in the Asteraceae family, including genera such as Artemisia, Achillea, Matricaria, and Tanacetum [31].Te fndings of this study highlight F. cernua as a signifcant source of apigenin and other favonoids, suggesting potential medicinal implications and health benefts.However, further research is essential to explore the pharmacological activities and potential therapeutic applications of these polyphenolic compounds.In addition, comprehensive investigations are needed to understand their pharmacological properties and safety aspects, particularly concerning potential therapeutic applications.
In addition to exploring the therapeutic potential of medicinal plants, assessing their potential toxic efects is also essential, as the negative impact on public health may remain unknown.In the present study, the acute toxicity of a polyphenolic extract of F. cernua was evaluated through  both in vitro and in vivo tests.Among these tests, the Artemia toxicity test was chosen due to the brine shrimp's sensitivity to toxic compounds, making it an excellent model for initial toxicity screening [32].Tis assay ofers numerous advantages, including cost-efectiveness, easy availability of commercial cysts, uniformity of the nauplii population after hatching, straightforward laboratory handling and maintenance, and the ability to perform assays in microplates [33].In addition, Artemia nauplii do not require feeding during the initial 72 h after hatching, making them highly sensitive to substances they come into contact with during this period [34].Te toxicity test with Artemia nauplii revealed that concentrations of 50 and 100 μg/ml of F. cernua extract were harmless to these invertebrates after 24 h of incubation.Furthermore, at a concentration of 1000 μg/ml, the maximum mortality observed was 14.4%, indicating that the lethal concentration of 50 (LC 50 ) is greater than 1000 μg/ml.An extract is generally considered nontoxic if its LC 50 value exceeds 1000 μg/ml [35].Terefore, the polyphenolic extract of F. cernua obtained in this study is considered nontoxic to Artemia salina.Considering the lack of existing reports on the toxicity of F. cernua toward Artemia or other aquatic invertebrates, the importance of investigating the toxicity of the extract in these organisms is highlighted.Tis is especially critical in the context of potential applications of this plant, such as its use as a biocide [10,18].Understanding the impact of F. cernua extract on aquatic organisms can help ensure its safe utilization in various applications, including agriculture.
Another in vitro test conducted was the hemolysis assay, which assesses the cytotoxicity of compounds or extracts by measuring erythrocyte rupture and hemoglobin release using spectrophotometry [36].Tis assay directly evaluates the potential damage that an extract may cause to the erythrocyte cell membrane.While this assay does not replicate the complex interactions within the human body, it provides valuable insight into the extract's potential impact on cell membranes [37].For this experiment, mouse and human erythrocytes were used along with two diferent vehicles for dissolving the extracts.In the case of mouse erythrocytes, the vehicle composed of Tween 80, DMSO, and PBS resulted in erythrocyte lysis from the frst hour.Saebo et al. reported that Tween 20 induces erythrocyte lysis at concentrations of ≥0.01%.Notably, human erythrocytes exhibited two times less lysis compared to mouse and rat erythrocytes [38].In this study, a fnal concentration of 0.1% Tween 80 was used, causing erythrocyte lysis; hence, a vehicle without Tween 80 was employed.Te DMSO: PBS vehicle did not cause any erythrocyte lysis during the 4h incubation period.Moreover, at a concentration of 500 μg/ ml, the polyphenolic extract of F. cernua exhibited hemolysis after 3 h of incubation, reaching a maximum value of 7.0 ± 3.1%, which is considered nontoxic [39].Regarding the hemolytic activity of the F. cernua extract on human erythrocytes, two vehicles were also used.Te vehicle composed of Tween 80, DMSO, and PBS resulted in hemolysis after the third hour of incubation, reaching a maximum hemolysis percentage of 56.4 ± 1.1 at 4 h of incubation.Te hemolytic efect was attributed to Tween 80, a nonionic detergent [40].Detergents possess a similar structure to the lipids in the plasma membrane, allowing them to penetrate the membrane and cause a reorganization of lipids and membrane proteins, ultimately leading to changes in erythrocyte morphology and lysis [41].Interestingly, the F. cernua extract showed lower hemolysis compared to the vehicle, possibly due to the presence of polyphenols, known for their cytoprotective efect on erythrocytes against oxidizing agents and hypotonic conditions [42,43].Te potential mechanism involves the polyphenols of the extract of F. cernua and their interaction with the polar heads of the membrane lipids, forming a barrier that prevents the detergent from interacting with the plasmatic membrane [44].In addition, the cytoprotective efect of apigenin on human erythrocytes subjected to oxidative damage by H 2 O 2 has been reported.Te proposed mechanism is that apigenin could restore cell surface morphology by protecting the sulfhydryl content in the red cell membrane protein [45].Tis cytoprotective efect was more noticeable at a concentration of 100 μg/ml, as higher concentrations resulted in increased hemolysis.In addition, the DMSO: PBS vehicle did not cause any damage to erythrocytes at any incubation hour.Similarly, the polyphenolic extract of F. cernua caused hemolysis only at a concentration of 500 μg/ml, reaching a value of 28.7 ± 1.2% at the fourth hour.Tis percentage, considered slightly toxic, was observed during the last hour of incubation [39].Extending the incubation time aimed to confrm that polyphenolic compounds would not afect the erythrocyte membrane, as certain compounds might require more time to interact and induce hemolysis [38].Tis is evident in the results since the analysis after only one hour of incubation would not have shown hemolysis in any of the treatments with the F. cernua extract.Tese results highlight the potential therapeutic value of the extract of F. cernua due to its cytoprotective properties and low hemolytic activity.
In the acute oral toxicity test, no deaths occurred among tested animals at doses of 300 and 2000 mg/kg.According to the OECD criteria, the LD 50 of the F. cernua extract is >2000-5000 mg/kg, categorized as category 5, and can be considered safe [46].However, some signs that could indicate slight intoxication, such as lethargy, drowsiness, and piloerection, were observed within the frst hours after administering the F. cernua extract.In addition, mice showed signifcant weight loss during the initial two days following the administration of the extract at a dose of 2000 mg/kg.Tese fndings align with a study by Zavala et al. [16], where an ethanolic extract at 2000 mg/kg did not cause death in Wistar rats.However, slight signs of toxicity, such as lethargy and drowsiness, were observed, although no diferences were found in terms of body weight.In contrast, in the present study, a decrease in animal weight was observed at the dose of 2000 mg/kg.Furthermore, the histopathological analysis revealed no alterations in liver and brain tissue.However, in relation to the kidney, observations showed some shrunken and smaller glomeruli, indicating possible kidney damage after administration of the 2000 mg/ kg dose.It is noteworthy that the toxicity of F. cernua appears to vary depending on the part of the plant, as dried Journal of Toxicology fruit has been reported to be toxic to cattle, causing infammation, ulcerations, and perforation of the gastrointestinal tract within the frst 24 h after consumption [47].In contrast, leaf extracts do not appear to have any lethal efect after acute consumption.However, there are reports suggesting that prolonged consumption may negatively impact liver function [15].Terefore, one of the perspectives is to evaluate the subacute and chronic efects that consumption of F. cernua extracts could have on liver and kidney function to determine whether prolonged consumption is safe for consumers.

Conclusions
In this study, a polyphenolic extract of Flourensia cernua was obtained using a novel ultrasound and microwave extraction method, followed by purifcation through ion-exchange chromatography.Analysis revealed its richness in favonoids and hydroxycinnamic acids, with apigenin and its glycosides being the most abundant compounds.Te extract showed no toxicity to Artemia salina and a low hemolytic efect on erythrocytes.In the acute oral toxicity test in mice, doses of 300 mg/kg and 2000 mg/kg were administered, and neither dose resulted in mortality among the animals.Tese fndings place the extract in category 5 according to the OECD standards, indicating that it is safe at tested concentrations.However, signs of potential toxicity, such as weight loss and kidney structural alterations, were observed at the highest dose tested.Terefore, further research is crucial to assess the safety profle of the extract under chronic exposure conditions and to evaluate its impact on liver and kidney functions following oral administration.

Figure 2 :Figure 3 :
Figure2: Efect of polyphenolic extract of F. cernua on the mortality of brine shrimp.Te diferent treatments are compared against the vehicle.Also, there is a signifcant diference between the 1000 μg/ml compared to the other doses.Ten brine shrimp per group were used in triplicate, and the experiment was conducted in three independent assays.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.Data are presented as mean ± SEM. ns: not signifcant.* * * p ≤ 0.001.

Figure 4 :FFigure 5 :
Figure4: Hemolysis of human red blood cells treated with the polyphenolic extract of F. cernua dissolved in Tween 80: DMSO: PBS (5 : 5 : 90) at diferent concentrations over various incubation times.No signifcant hemolysis was observed at 1 and 2 hours of incubation compared to the vehicle.However, the vehicle itself caused signifcant hemolysis starting at 3 hours of incubation.Te extracts showed a cytoprotective efect by reducing the hemolysis induced by the vehicle, with this efect being more noticeable at the fourth hour of incubation at the concentration of 100 μg/ml compared to the other concentrations used.Each treatment was performed in triplicate in two independent experiments.Statistical analysis was performed using a two-way ANOVA followed by Tukey's post hoc test.Data are presented as mean ± SEM. ns: not signifcant.* * * p ≤ 0.001.

Figure 6 :
Figure 6: Weight of the mice subjected to the acute oral toxicity test.(a) Weight of the mice at diferent times after administration of polyphenolic extract of F. cernua.(b) Delta weight compared to the initial weight.N � 6 mice per group.Statistical analysis was performed using a two-way ANOVA followed by Tukey's post hoc test.Data are presented as mean ± SEM. ns: not signifcant.* p ≤ 0.05.

Figure 7 :
Figure 7: Photomicrographs of hematoxylin and eosin (H&E)-stained sections of the liver, kidney, and brain from mice treated with vehicle and the polyphenolic extract of F. cernua (300 and 2000 mg/kg).Black arrows indicate shrunken glomeruli in mice that received the 2000 mg/kg dose.Scale bar: 50 μm.
10)2.10.Acute Oral Toxicity According to OECD 423.Te study was performed after getting approval from the Ethics Committee of the School of Chemistry, Autonomous University of Coahuila with the registration number: P-FCQ-A-25-01-23-1.Te acute oral toxicity test was performed following the guidelines of the OECD (Organization for Economic Co-operation and Development) 423 assay.Female mice (Mus musculus) of the BALB/c strain, weighing between 18 and 22 g and aged 8 to 12 weeks, were used for the study.According to the OECD guidelines, females are normally used because they generally exhibit slightly higher sensitivity to toxic efects.Tis ensures a more sensitive and reliable assessment of the potential toxicity of the substance being tested.Animals were housed and handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no.80-23, revised 1978) and the Mexican normative NOM-062-ZOO-1999.Mice were kept under controlled conditions with 12/12 h light/dark cycles, 22 ± 2 °C, 40-60% relative humidity, with water and food (LabDiet 5001 Rodent Diet) available ad libitum.

Table 1 :
Polyphenol content of the polyphenolic-enriched extract of F. cernua.
3.5.Histopathological Analysis of Liver, Kidney, and Brain.Histological examination of the liver, kidney, and brain tissues of mice was conducted to assess any potential alterations caused by the administration of F. cernua extract (Figure7).Liver tissues from all experimental groups, including the vehicle group and both doses of the F. cernua extract, exhibited a normal hepatocellular architecture.Observations showed intact central veins and trabeculae without any histological abnormalities detected in liver tissue.Regarding the kidneys, both the vehicle group and the 300 mg/kg dose group of F. cernua extract displayed normal

Table 2 :
Main polyphenolic compounds in the polyphenolic extract of F. cernua.